Glycosylation and the Demands of Antibody Engineering

Engineered antibodies are the quintessential success story of the biopharmaceutical industry today. Several approved antibodies have significantly advanced the treatment of cancers and autoimmune diseases and are currently garnering immense profits for their parent companies. It is estimated that approximately 30% of new drugs likely to achieve approval in the next decade will be antibodies or antibody fragments, and the number may run even higher as engineering technologies improve.

Improvements in analytical technology and identification of subtle modifications in glycosylation patterns have created a demand for a higher level of understanding defining the interactive mechanisms of these molecules with their targets.

A leading figure in the study of glycosylation is Roy Jefferis, PhD, of the University of Birmingham, UK. Armed with formidable analytical tools, Jefferis and his colleagues are now able to ask extremely detailed and precise questions concerning how sugars drive the functions of antibodies and how they affect their performance as therapeutics.

In a recent interview, Jefferis elaborated on some of the themes presented in his extensive reviews on the art and science of antibody glycosylation.

KJM: Antibodies and other proteins are perhaps the most expensive drugs on the market. How will technical advances in glycosylation and the legal wrangling over their intellectual property rights affect their pricing?

RJ: Antibodies developed for treatments in cancer depend on: 1) specificity for antigens expressed on the tumor cells and 2) effector functions that destroy the target cell by activating downstream biological reactions. Protein and glycosylation engineering is employed to generate antibodies with enhanced effector functions. The presence or absence of one sugar residue—fucose—can result in a two-orders-of-magnitude difference in the ability to kill cancer cells by antibody-dependent cell cytotoxicity (ADCC). A consequent impact on dose, and hence cost, is anticipated.

KJM: Recombinant antibodies have proven to be immunogenic, at least in a proportion of patients. What properties influence immunogenicity?

Quick Recap

RJ: Rituxan and Herceptin are not fully human antibodies—the region conferring specificity is of mouse origin—so it's not surprising that they raise an immune response in certain cases. But even fully human antibodies can raise an immune response. A very important factor in their production is that the preparations be aggregate-free. This puts a lot of pressure on downstream quality control, since a bad batch could immunize a patient for life, and exclude him as a candidate for this therapy. Glycosylation can enhance the solubility of protein molecules and may protect against aggregation.

KJM: Is IgG1 always the best choice for a therapeutic antibody? What role does glycosylation play in that decision?

RJ: No, it depends on the task that you have selected—i.e., the disease indication. In oncology, we want to kill cells, and an IgG1 antibody bearing non-fucosylated oligosaccharides may be optimal. For other jobs a Fab fragment may be sufficient. For example, an anti-tumor necrosis factor Fab fragment has clinical efficacy; it is pegylated to confer an extended half-life.

KJM: Can one reliably extrapolate performance from in vitro studies and in vivo animal models to humans?

RJ: No, definitely not! FDA insists on animal data before investigators can move ahead with human trials, but simply because the drug appears benign in animal trials, that still doesn't mean that it's entirely safe. Improved animal models are being developed using genetic engineering, e.g., mice and other animals in which endogenous Fc receptors are "knocked out" and human Fc receptors are "knocked in" to the genome. This provides a more realistic model, but in the final analysis, extensive human trials are essential, including Phase 4 monitoring.